The use of ‘so-called’ protein scaffolds is gaining attention in biochemistry as a potential route to generating novel ligand binding proteins for use in research and medicine. Such scaffold proteins, used to display one or more peptide sequences, can potentially provide an alternative to antibodies or antibody fragments.
The term ‘protein scaffold’ is used to describe a type of polypeptide structure that is observed in differing contexts and with distinct biochemical functions. Because of their intrinsic conformational stability it has been reasoned that such scaffolds might be amenable to protein engineering.
It is conventionally thought that immunoglobulins (antibodies) owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of hypervariable peptide segments, these segments are variable both in sequence and in conformation. After antibody engineering methods along with library techniques resulted in successes in the selection of functional antibody fragments, interest began to grow in using other protein architectures to synthesise useful binding proteins.
Descriptions of the desirable properties of a suitable protein scaffold taken include the following;
“Candidates for suitable protein scaffolds ought to exhibit a compact and structurally rigid core that is able to present surface loops of varying sequence and length or to otherwise tolerate side chain replacements in a contiguous surface region, including exposed hydrophobic residues, without significant changes in their folding properties.” Skerra A: Engineered protein scaffolds for molecular recognition. J Mol Recognit 2000, 13:167-187 and The term ‘scaffold’, as used in protein engineering, describes a single chain polypeptidic framework typically of reduced size (<200 AA) and containing a highly structured core associated with variable portions of high conformational tolerance allowing insertions, deletions, or other substitutions'. Wurch et al., Trends in Biotechnology, November 2012, 30, 575-582.
However, not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand binding sites with high affinities and specificities.
Prior art scaffolds include inactivated staphylococcal nuclease, green fluorescent protein (GFP) and thioredoxin A (TrxA), the fibronectin type III domain (‘Fn3’), lipocalin family proteins, bilin binding protein (BBP), as well as isolated protein folds such as the Z domain of staphylococcal protein A, “affibodies”, anticafins, and ankyrin repeats, and others.
WO 2006/131749 describes several rational mutations made in Stefin A to improve it as a scaffold. The modified Stefin A scaffold comprises mutations at the following three sites Lys71-Leu73, V48D and G4W and is referred to as STM (Stefin A Triple Mutant).
WO2009/136182 describes further refinements of STM scaffolds.
There remains a need for improved scaffold proteins. In particular, it has been found that prior art scaffolds are often unable to sufficiently stabilise peptide aptamers, and the presence of the aptamers in the scaffold actually causes the scaffold to significantly deform. See for example Woodman et al., ‘Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers’, J. Mol. Biol. (2005) 352, 1118-1133
Ideally such improved scaffold proteins would provide one or more of the following benefits:                Improved stability to provide a rigid framework that does not deform;        Smaller size;        Improved ability to support high quality and complexity libraries;        Improved affinity of selected binding proteins for their target; and        Simplicity of further manipulation due to accessible N- and C-terminal ends.        